The genetic diversity of 221 Mimosa pudica bacterial symbionts trapped from eight soils from diverse environments in French Guiana was assessed by 16S rRNA PCR-RFLP, REP-PCR fingerprints, as well as by phylogenies of their 16S rRNA and recA housekeeping genes, and by their nifH, nodA and nodC symbiotic genes. Interestingly, we found a large diversity of beta-rhizobia, with Burkholderia phymatum and Burkholderia tuberum being the most frequent and diverse symbiotic species. Other species were also found, such as Burkholderia mimosarum, an unnamed Burkholderia species and, for the first time in South America, Cupriavidus taiwanensis. The sampling site had a strong influence on the diversity of the symbionts sampled, and the specific distributions of symbiotic populations between the soils were related to soil composition in some cases. Some alpha-rhizobial strains taxonomically close to Rhizobium endophyticum were also trapped in one soil, and these carried two copies of the nodA gene, a feature not previously reported. Phylogenies of nodA, nodC and nifH genes showed a monophyly of symbiotic genes for beta-rhizobia isolated from Mimosa spp., indicative of a long history of interaction between beta-rhizobia and Mimosa species. Based on their symbiotic gene phylogenies and legume hosts, B. tuberum was shown to contain two large biovars: one specific to the mimosoid genus Mimosa and one to South African papilionoid legumes.

Nutritional symbiotic interactions require the housing of large numbers of microbial symbionts, which produce essential compounds for the growth of the host. In the legume-rhizobium nitrogen-fixing symbiosis, thousands of rhizobium microsymbionts, called bacteroids, are confined intracellularly within highly specialized symbiotic host cells. In Inverted Repeat-Lacking Clade (IRLC) legumes such as Medicago spp., the bacteroids are kept under control by an arsenal of nodulespecific cysteine-rich (NCR) peptides, which induce the bacteria in an irreversible, strongly elongated, and polyploid state. Here, we show that in Aeschynomene spp. legumes belonging to the more ancient Dalbergioid lineage, bacteroids are elongated or spherical depending on the Aeschynomene spp. and that these bacteroids are terminally differentiated and polyploid, similar to bacteroids in IRLC legumes. Transcriptome, in situ hybridization, and proteome analyses demonstrated that the symbiotic cells in the Aeschynomene spp. nodules produce a large diversity of NCR-like peptides, which are transported to the bacteroids. Blocking NCR transport by RNA interference-mediated inactivation of the secretory pathway inhibits bacteroid differentiation. Together, our results support the view that bacteroid differentiation in the Dalbergioid clade, which likely evolved independently from the bacteroid differentiation in the IRLC clade, is based on very similar mechanisms used by IRLC legumes.

We investigated the presence of endophytic rhizobia within the roots of the wetland wild rice Oryza breviligulata, which is the ancestor of the African cultivated rice Oryza glaberrima. This primitive rice species grows in the same wetland sites as Aeschynomene sensitiva, an aquatic stem-nodulated legume associated with photosynthetic strains of Bradyrhizobium. Twenty endophytic and aquatic isolates were obtained at three different sites in West Africa (Senegal and Guinea) from nodal roots of O. breviligulata and surrounding water by using A. sensitiva as a trap legume. Most endophytic and aquatic isolates were photosynthetic and belonged to the same phylogenetic Bradyrhizobium/Blastobacter subgroup as the typical photosynthetic Bradyrhizobium strains previously isolated from Aeschynomene stem nodules. Nitrogen-fixing activity, measured by acetylene reduction, was detected in rice plants inoculated with endophytic isolates. A 20% increase in the shoot growth and grain yield of O. breviligulata grown in a greenhouse was also observed upon inoculation with one endophytic strain and one Aeschynomene photosynthetic strain. The photosynthetic Bradyrhizobium sp. strain ORS278 extensively colonized the root surface, followed by intercellular, and rarely intracellular, bacterial invasion of the rice roots, which was determined with a lacZ-tagged mutant of ORS278. The discovery that photosynthetic Bradyrhizobium strains, which are usually known to induce nitrogen-fixing nodules on stems of the legume Aeschynomene, are also natural true endophytes of the primitive rice O. breviligulata could significantly enhance cultivated rice production.

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The occurrence of alternative Nod factor (NF)-independent symbiosis between legumes and rhizobia was first demonstrated in some Aeschynomene species that are nodulated by photosynthetic bradyrhizobia lacking the canonical nodABC genes. In this study, we revealed that a large diversity of non-photosynthetic bradyrhizobia, including B. elkanii, was also able to induce nodules on the NFindependent Aeschynomene species, A. indica. Using cytological analysis of the nodules and the nitrogenase enzyme activity as markers, a gradient in the symbiotic interaction between bradyrhizobial strains and A. indica could be distinguished. This ranged from strains that induced nodules that were only infected intercellularly to rhizobial strains that formed nodules in which the host cells were invaded intracellularly and that displayed a weak nitrogenase activity. In all nonphotosynthetic bradyrhizobia, the type III secretion system (T3SS) appears required to trigger nodule organogenesis. In contrast, genome sequence analysis revealed that apart from a few exceptions, like the Bradyrhizobium ORS285 strain, photosynthetic bradyrhizobia strains lack a T3SS. Furthermore, analysis of the symbiotic properties of an ORS285 T3SS mutant revealed that the T3SS could have a positive or negative role for the interaction with NF-dependent Aeschynomene species, but that it is dispensable for the interaction with all NF-independent Aeschynomene species tested. Taken together, these data indicate that two NF-independent symbiotic processes are possible between legumes and rhizobia: one dependent on a T3SS and one using a so far unknown mechanism.

Rhizobia are soil bacteria able to develop a nitrogen-fixing symbiosis with legumes. They are taxonomically spread among the alpha and beta subclasses of the Proteobacteria. Mimosa pudica, a tropical invasive weed, has been found to have an affinity for beta-rhizobia, including species within the Burkholderia and Cupriavidus genera. In this study, we describe the diversity of M. pudica symbionts in the island of New Caledonia, which is characterized by soils with high heavy metal content, especially of Ni. By using a plant-trapping approach on four soils, we isolated 96 strains, the great majority of which belonged to the species Cupriavidus taiwanensis (16S rRNA and recA gene phylogenies). A few Rhizobium strains in the newly described species Rhizobium mesoamericanum were also isolated. The housekeeping and nod gene phylogenies supported the hypothesis of the arrival of the C. taiwanensis and R. mesoamericanum strains together with their host at the time of the introduction of M. pudica in New Caledonia (NC) for its use as a fodder. The C. taiwanensis strains exhibited various tolerances to Ni, Zn and Cr, suggesting their adaptation to the specific environments in NC. Specific metal tolerance marker genes were found in the genomes of these symbionts, and their origin was investigated by phylogenetic analyses.

A better understanding of how bacteria resist stresses encountered during the progression of plant-microbe symbioses will advance our ability to stimulate plant growth. Here, we show that the symbiotic system comprising the nitrogen-fixing bacterium Bradyrhizobium diazoefficiens and the legume Aeschynomene afraspera requires hopanoid production for optimal fitness. While methylated (2Me) hopanoids contribute to growth under plant-cell-like microaerobic and acidic conditions in the free-living state, they are dispensable during symbiosis. In contrast, synthesis of extended (C35) hopanoids is required for growth microaerobically and under various stress conditions (high temperature, low pH, high osmolarity, bile salts, oxidative stress, and antimicrobial peptides) in the free-living state and also during symbiosis. These defects might be due to a less rigid membrane resulting from the absence of free or lipidA-bound C35 hopanoids or the accumulation of the C30 hopanoid diploptene. Our results also show that C35 hopanoids are necessary for symbiosis only with the host Aeschynomene afraspera but not with soybean. This difference is likely related to the presence of cysteine-rich antimicrobial peptides in Aeschynomene nodules that induce drastic modification in bacterial morphology and physiology. The study of hopanoid mutants in plant symbionts thus provides an opportunity to gain insight into host-microbe interactions during later stages of symbiotic progression, as well as the microenvironmental conditions for which hopanoids provide a fitness advantage.

Research on the nitrogen-fixing symbiosis has been focused, thus far, on two model legumes, Medicago truncatula and Lotus japonicus, which use a sophisticated infection process involving infection thread formation. However, in 25% of the legumes, the bacterial entry occurs more simply in an intercellular fashion. Among them, some Aeschynomene spp. are nodulated by photosynthetic Bradyrhizobium spp. that do not produce Nod factors. This interaction is believed to represent a living testimony of the ancestral state of the rhizobium-legume symbiosis. To decipher the mechanisms of this Nod-independent process, we propose Aeschynomene evenia as a model legume because it presents all the characteristics required for genetic and molecular analysis. It is a short-perennial and autogamous species, with a diploid and relatively small genome (2n=20; 460 Mb/1C). A. evenia 'IRFL6945' is nodulated by the well-characterized photosynthetic Bradyrhizobium sp. strain ORS278 and is efficiently transformed by Agrobacterium rhizogenes. Aeschynomene evenia is genetically homozygous but polymorphic accessions were found. A manual hybridization procedure has been set up, allowing directed crosses. Therefore, it should be relatively straightforward to unravel the molecular determinants of the Nod-independent process in A. evenia. This should shed new light on the evolution of rhizobium-legume symbiosis and could have important agronomic implications.